Truncated forms of NY-ESO-1 protein

ABSTRACT

The invention relates to antibodies which bind to the cancer associated antigen NY-ESO-1. Both polyclonal and monoclonal antibodies are part of the invention, as are chimeric forms of the antibodies, and binding portions of antibodies. Uses of these antibodies are described. Also described are truncated, recombinant forms of the cancer associated antigen.

RELATED APPLICATION

[0001] This application is a continuation-in-part of Ser. No.08/937,263, filed Sep. 15, 1997, which is a continuation-in-part of Ser.No. 08/725,182, filed Oct. 3, 1996 now U.S. Pat. No. ______. Both ofthese applications are incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to antibodies against an antigenassociated with cancer as well as the uses of these, as well as variantsof this protein, and HLA binding peptides derived from

BACKGROUND AND PRIOR ART

[0003] It is fairly well established that many pathological conditions,such as infections, cancer, autoimmune disorders, etc., arecharacterized by the inappropriate expression of certain molecules.These molecules thus serve as “markers” for a particular pathological orabnormal condition. Apart from their use as diagnostic “targets”, i.e.,materials to be identified to diagnose these abnormal conditions, themolecules serve as reagents which can be used to generate diagnosticand/or therapeutic agents. A by no means limiting example of this is theuse of cancer markers to produce antibodies specific to a particularmarker. Yet another non-limiting example is the use of a peptide whichcomplexes with an MHC molecule, to generate cytolytic T cells againstabnormal cells.

[0004] Preparation of such materials, of course, presupposes a source ofthe reagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

[0005] To date, two strategies have been employed for the detection ofsuch antigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines which are tested for theexpression of the specific antigen. The biochemical approach,exemplified by, e.g., O. Mandelboim, et al., Nature 369: 69 (1994)incorporated by reference, is based on acidic elution of peptides whichhave bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

[0006] These two approaches to the molecular definition of antigens havethe following disadvantages: first, they are enormously cumbersome,time-consuming and expensive; and second, they depend on theestablishment of cytotoxic T cell lines (CTLs) with predefinedspecificity.

[0007] The problems inherent to the two known approaches for theidentification and molecular definition of antigens is best demonstratedby the fact that both methods have, so far, succeeded in defining onlyvery few new antigens in human tumors. See, e.g., van der Bruggen etal., Science 254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178:489-495 (1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994);Kawakami, et al., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

[0008] Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer, associatedantigens.

[0009] One key methodology is described by Sahin, et al., Proc. Natl.Acad. Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also,see U.S. Pat. No. 5,698,396, and patent application Ser. No. 08/479,328filed Jan. 3, 1996. All three of these references are incorporated byreference. To summarize, the method involves the expression of cDNAlibraries in a prokaryotic host. (The libraries are secured from a tumorsample). The expressed libraries are then immnoscreened with absorbedand diluted sera, in order to detect those antigens which elicit hightiter humoral responses. This methodology is known as the SEREX method(“Serological identification of antigens by Recombinant ExpressionCloning”). The methodology has been employed to confirm expression ofpreviously identified tumor associated antigens, as well as to detectnew ones. See the above referenced patent applications and Sahin, etal., supra, as well as Crew, et al., EMBO J 144: 2333-2340 (1995).

[0010] The SEREX methodology has been applied to esophageal cancersamples, and an antigen has now been identified, and its encodingnucleic acid molecule isolated and cloned. This is the subject of theapplications which are incorporated by reference. The antigen andtruncated forms have been found to be reactive with antibodies in theserum of cancer patients. This, inter alia, is the subject of theinvention, which is described in more detail in the disclosure whichfollows.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 shows the expression pattern of RNA for the NY-ESO-1antigen, in various tissue types.

[0012]FIG. 2 shows Northern Blot analysis of NY-ESO-1 mRNA, which wasfound in testis and cell line SK-MEL-19, but not in various other celland tissue samples.

[0013]FIG. 3 shows potential sites for modification of the deduced aminoacid sequence of NY-ESO-1.

[0014]FIG. 4 is a hydrophilicity plot of NY-ESO-1, showing hydrophilicdomains in the amino terminus and a long, hydrophobic stretch close tothe carboxyl end.

[0015]FIG. 5 shows the results of CTL lysis studies using various cellswhich are HLA-A2 positive, NY-ESO-1 positive, positive for both, orpositive for neither.

[0016]FIG. 6 presents data establishing that HLA-A2 is the presentingmolecule for presentation of SEQ ID NO: 1 derived peptides.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0017] Total RNA was extracted from a snap frozen specimen of well tomoderately differentiated squamous cell cancer of the esophagus, usingwell known methods. See, e.g., Chomzynski, J. Analyt. Biochem. 162:156-159 (1987), for one such method. This RNA was used to prepare a cDNAlibrary which was then transfected into λZAP phage vectors, inaccordance with the manufacturer's instructions. The λZAP library wasthen transfected into E. coli, yielding 1.6×10⁶ primary isolates.

[0018] The SEREX methodology of Sahin, et al., Proc. Natl. Acad. Sci.USA 92: 11810-11813 (1995), incorporated by reference, was then used. Inbrief, autologous serum was stripped of antibodies against moleculeswhich are endogenous to E. coli by combining the serum with lysates ofE. coli transfected with phage λZAP which did not contain the cDNAclones from the esophageal cancer cells.

[0019] The depleted serum was then diluted, and mixed withnitrocellulose membranes containing phage plaques. The plaques wereincubated overnight, at room temperature. Washing followed, and then thefilters were incubated with alkaline phosphatase conjugated goat antihuman FCγ secondary antibodies, and reactive phage plaques werevisualized by incubating with 5-bromo-4-chloro-indolyl phosphate andnitroblue tetrazolium. A total of 13 positive clones were found.

EXAMPLE 2

[0020] Following identification, the reactive clones were subcloned tomonoclonality via dilution cloning and testing with human serum. Theseclones were then purified, excised in vitro, and converted into pBK-CMVplasmid forms using the manufacturer's instructions. The inserted DNAwas then evaluated using EcoRI-XbaI restriction mapping to determinedifferent inserts. Eight different inserts were identified, ranging insize from about 500 to about 1.3 kilobase pairs. The clones weresequenced using an ABI PRISM automated sequencer.

[0021] Table 1 summarizes the results. One gene was represented by fouroverlapping clones, a second by three overlapping clones, and theremaining six by one clone only.

[0022] A homology search revealed that the clones referred to asNY-ESO-2, 3, 6, 7 were already known. See Elisei, et al., J. Endocrin.Invest. 16: 533-540 (1993); Spritz, et al., Nucl. Acids Res. 15:10373-10391 (1987); Rabbits, et al., Nature Genetics 4: 175-180 (1993);Crozat, et al., Nature 363: 640-644 (1993); GenBank H18368 and D25606.Two of the clones (NY-ESO-3 and NY-ESO-6), have previously been shown tobe expressed in various normal human tissues. No evidence of lineagerestriction has been found. NY-ESO-6 (cDNA), appears to be the3′-untranslated portion of the FUS/TLS gene. In experiments not reportedhere, sequencing and Southern Blot analysis of NY-ESO-6 showed noevidence of translocation or point mutations in the cancer. Four of theclones, i.e., NY-ESO-1, 4, 5 and 8 showed no strong homology tosequences in the databases examined, and were thus studied further.TABLE 1 Genes isolated from esophageal cancer library by immunoscreeningwith autologous serum GENE CLONE# Size DNA databank Comments NY-ESO-1E1-5b 679 bp No strong expressed in testis E1-114b 614 bp homology andovary E1-153c 670 bp E1-50 679 bp NY-ESO-2 E1-71a 605 bp U1 small clonedby Ab E1-140 874 bp nuclear RNP 1 screening E1-31 750 bp homolog(thyroiditis patient) NY-ESO-3 E1-141b 517 bp Colon 3′ direct (dbjD25606, MboI cDNA; gb H18638) Adult brain unpublished cDNA NY-ESO-4E1A-10c 400 bp No strong ubiquitous homology expression in normaltissues NY-ESO-5 E1A-54 670 bp No strong expressed in normal homologyesophagus NY-ESO-6 E1B-9b −1.2 kb Human fus translocated in mRNAliposarcoma t (12; 16) NY-ESO-7 E1B-20f −1.0 kb human U1-70k differentfrom sn RNP NY-ESO-2 (embl HSU17052, gbM22636) NY-ESO-8 E1B-20g −1.3 kbNo strong ubiquitous homology expression in normal tissues

EXAMPLE 3

[0023] Studies were carried out to evaluate mRNA expression of theNY-ESO 1, 4, 5 and 8 clones. To do this, specific oligonucleotideprimers were designed for each sequence, such that cDNA segments of300-400 base pairs could be amplified, and so that the primer

[0024] melting temperature would be in the range of 65-70° C. Reversetranscription-PCR was then carried out using commercially availablematerials and standard protocols. A variety of normal and tumor celltypes were tested. The clones NY-ESO-4 and NY-ESO-8 were ubiquitous, andwere not studied further. NY-ESO-5 showed high level expression in theoriginal tumor, and in normal esophageal tissue, suggesting that it wasa differentiation marker.

[0025] NY-ESO-1 was found to be expressed in tumor mRNA and in testis,but not normal colon, kidney, liver or brain tissue. This pattern ofexpression is consistent with other tumor rejection antigen precursors.

EXAMPLE 4

[0026] The RT-PCR assay set forth supra was carried out for NY-ESO-1over a much more complete set of normal and tumor tissues. Tables 2, 3and 4 show these results. In brief, NY-ESO-1 was found to be highlyexpressed in normal testis and ovary cells. Small amounts of RT-PCRproduction were found in normal uterine myometrium, and not endometrium,but the positive showing was not consistent. Squamous epithelium ofvarious cell types, including normal esophagus and skin, were alsonegative.

[0027] When tumors of unrelated cell lineage were tested, 2 of 11melanomas cell lines showed strong expression, as did 16 of 67 melanomaspecimens, 6 of 33 breast cancer specimens and 4 of 4 bladder cancer.There was sporadic expression in other tumor types. TABLE 2 mRNAdistribution of NY-ESO-1 in normal tissues Tissue mRNA Tissue mRNAEsophagus − Adrenal − Brain* − Pancreas − Fetal Brain − Seminal Vesicle− Heart − Placenta − Lung − Thymus − Liver − Lymph node − Spleen −Tonsil − Kidney − PBL − Stomach − PBL, activated# − Small intestine −Melanocytes − Colon − Thyroid − Rectum − Uterus +/−** Breast − Testis +Skin − Ovary +

[0028] TABLE 3 mRNA distribution of NY-ESO-1 in melanoma and breastcancer cell lines: Cell line NY-ESO-1 mRNA MZ2-MEL3.1 − MZ2-MEL2.2 −SK-MEL-13 − SK-MEL-19 + SK-MEL-23 − SK-MEL-29 − SK-MEL-30 − SK-MEL-31 −SK-MEL-33 − SK-MEL-37 + SK-MEL-179 − SK-BR-3 − SK-BR-5 − 734B −MDA-MB-231 −

[0029] TABLE 4 NY-ESO-1 mRNA expression in various human tumors byRT-PCR mRNA mRNA tumor type (positive/total) tumor type (positive/total)melanoma 25/77  ovarian cancer 2/8 breast cancer 17/43  thyroid cancer2/5 prostate cancer 4/16 bladder cancer  9/13 colon cancer 0/16Burkitt's lymphoma 1/2 glioma 0/15 basal cell carcinoma 0/2 gastriccancer 0/12 Jejomyosarcoma 0/2 lung cancer 5/17 other sarcomas 0/2 renalcancer 0/10 pancreatic cancer 0/2 lymphoma* 0/10 seminoma 0/1 hepatoma2/7  spinal cord tumor 0/1

[0030] A further set of experiments were carried out to ascertain if thepresence of anti NY-ESO-1 antibody in cancer patient sera could bedetermined via an ELISA.

[0031] To elaborate, recombinant NY-ESO-1 in a solution of coatingbuffer (15 mM Na₂CO₃, 30 mM NaHCO₃, pH 9.6, 0.02% NaN₃), at aconcentration of 1 ug/ml, was adsorbed to microwell plates (10 ul ofsolution per well), and then kept overnight at 4° C. The plates werewashed with phosphate buffered saline, and blocked, overnight, at 4° C.,with 10 ul/well of 2% bovine serum albumin/phosphate buffered saline.After washing, 10 ul/well of diluted serum in 2% bovine serum albuminwas added to the wells. Following two hours of incubation at roomtemperature, plates were washed, and 10 ul/well of goat anti-humanIgG-alkaline phosphatase conjugates were added, at a 1:1500 dilution.This solution was incubated for one hour at room temperature, followedby washing and addition of a solution of substrate for the alkalinephosphatase (10 ul/well). After 25 minutes at room temperature, thewells were read with a fluorescence plate reader. The results arepresented in the following table: Cancer patients: Eso 1 +/total tested% melanoma 12/127 9.4 ovarian cancer 4/32 12.5 lung cancer 1/24 4.0breast cancer 2/26 7.7 Blood donors 0/70 0

[0032] In order to determine whether there was a relationship betweenexpression of mRNA for NY-ESO-1 in tumors, and antibody response to theNY-ESO-1 protein, data from sixty-two melanoma patients were compared.All patients whose serum was reactive with NY-ESO-1 protein (i.e.,contained antibodies to NY-ESO-1), also had NY-ESO-1 positive tumors,while no patients with NY-ESO-1 negative tumors showed antibodies toNY-ESO-1 in their serum. There was a percentage of NY-ESO-1 positivepatients who lacked the antibody. Given that about 20-40% of melanomasexpressed NY-ESO-1, and only patients with NY-ESO-1 positive tumors haveantibody, the data suggest a high percentage of patients with NY-ESO-1positive tumors develops antibodies against the protein, thus suggestinga broad scale assay useful in diagnosis and responsiveness to treatment.

EXAMPLE 5

[0033] Northern blot analysis was then carried out to investigate thesize of the NY-ESO-1 transcript, and to confirm tissue expressionpatterns. The methodology of Ausubel, et al., Current Protocols InMolecular Biology (John Wiley & Sons, 1995) was used. To be specific, 20ug of total RNA per lane were dissolved in a formamide and formaldehydecontaining buffer; heated to 65° C., and then separated on a 1.2%agarose gel, with 3% formaldehyde, followed by transfer tonitrocellulose paper. Hybridization was then carried out using a ³²Plabelled probe, followed by high stringency washing. The final wash wasat 0.1×SSC, 0.1% SDS, 60° C., for 15 minutes.

[0034] RNA from testis, and a melanoma cell line (SK-MEL-19) which hadbeen positive for NY-ESO-1 in the prior assays, showed an RNA transcriptof about 0.8-0.9 kb. An esophageal carcinoma specimen showed a smear inthe 0.4-0.9 kb range, reflecting partial degradation. RNA fromadditional tissues or cell lines tested showed no transcript.

[0035] To get cDNA encoding the full transcript, the esophageal cDNAlibrary was rescreened, using plaque hybridization, and the originalcDNA clone as the hybridization probe. When 3×10⁵ clones were screened,six positives were found. The three longest clones were sequenced.Analysis of open reading frames showed that all three contained theentire coding region, and 5′-untranslated regions of variable size. Thelongest clone, 755 base pairs in length, (excluding polyA), contains a543 base pair coding region, together with 53 untranslated bases at the5′ end and 151 untranslated base pairs at the 3′-end. See SEQ ID NO: 1(also, FIG. 3).

[0036] The long ORF indicated that the deduced sequence of NY-ESO-1protein is 180 amino acids. The single immunopositive clone contained asequence encoding 173 of these. Deduced molecular mass is 17,995daltons.

[0037] Analysis shows that there is an abundance of glycine residues inthe N-terminal portion (30 of the first 80, 4 in the remaining 100).Hydrophilicity analysis indicated that there were hydrophilic antigenicsequences in the N-terminal half of the molecule, with alternatinghydrophobic and hydrophilic sequences, ending with a long, C-terminalhydrophobic tail (amino acids 152-172), followed by a short hydrophilictail. This pattern suggests a transmembrane domain. There are severalpotential N-myristorylation sites, 3 phosphorylation sites, and noevidence of N-glycosylation sites

EXAMPLE 6

[0038] A melanoma cell line “NW-MEL-38” was established, in 1995, from apatient who suffered from malignant melanoma. Serum samples, peripheralblood lymphocytes, and tumor samples, were taken from the subject andfrozen, until the work described herein was carried out. In anticipationof evaluating antitumor T cell response in this patient, the patient wasHLA typed as HLA-A1 and HLA-A2.

[0039] To determine whether melanoma from this patient expressedNY-ESO-1, total RNA was isolated from both tumor samples and cell lineNW-MEL-38, using standard techniques. Then, two micrograms of the totalRNA, from each samples were subjected to cDNA synthesis, again usingstandard techniques.

[0040] The cDNA was then used in RT-PCR experiments, using the followingprimers: 5′-CACACAGGAT CCATGGATGC TGCAGATGCG G′-3′ (SEQ ID NO: 2), andCACACAAAGC TTGGCTTAGC GCCTCTGCCC TG-3′ (SEQ ID NO: 3)

[0041] These primers should amplify a segment of SEQ ID NO: 1 whichspans nucleotides 271 to 599.

[0042] Amplification was carried out over 35 cycles, using an annealingtemperature of 60° C. The PCR products were visualized via ethidiumbromide staining, on a 1.5% agarose gel.

[0043] The results indicated that both the tumor and the cell lineexpressed SEQ ID NO: 1. The cell line and tumor samples were used insubsequent experiments.

EXAMPLE 7

[0044] The isolated cDNA molecule, discussed supra, was then used tomake recombinant protein. Specifically, the cDNA was PCR amplified,using standard techniques, and was then cloned into a commerciallyavailable plasmid vector, i.e., pQE9, which contains His tags. In worknot elaborated upon herein, a second vector, pQE9K was also used. Thisdiffers from PQE9 in that kanamycin resistance is imparted by pQE9K,rather than ampicillin resistance.

[0045] The plasmid vector was transformed into E. coli strain XL1-Blue,and positive transformants were identified via restriction mapping andDNA sequencing. Production of recombinant protein was induced usingisopropyl β-D-thiogalactoside, and the protein was purified on an Ni²⁺ion chromatography column, following well known procedures. The proteinwhen analyzed via 15% SDS-PAGE and silver staining, was identified as aprotein with a molecular weight of about 22 kilodaltons. This isconsistent with the anticipated size of the protein from its sequence.Two other forms of the recombinant protein were also identified. Theseconsisted of amino acids 10-180, and 10-121 of the amino acid sequencereported in SEQ ID NO: 1. They have molecular weights of about 14 kD and20 kD, respectively, on SDS-PAGE, as carried out supra.

[0046] An additional set of experiments were carried out to expressNY-ESO-1 in baculovirus. To elaborate, the NY-ESO-1 cDNA insert wasreleased from the pQE9 vector, by cleavage with BamHI and HindIII. Thisinsert was then subcloned into a commercially available baculovirusvector which had been cleaved with the same enzymes. Positive cloneswere determined, using standard methods, and transfected into recipientSf9 cells. Recombinant viruses were then used to infect insect cells,using a standard medium (IPL-41), supplemented with 10% fetal calfserum. The multiplicity of infection for the work was 20. Expression ofrecombinant protein was determined as described supra. The recombinantprotein produced in this vector carries an His-tag, so it was purifiedon Ni²⁺ affinity columns, also as described, supra. The protein consistsof amino acids 10-180, and has a molecular weight of 20 kD via SDS-PAGE.

[0047] Additional eukaryotic transfectants were then produced. To dothis, the NY-ESO-1 coding sequence was isolated from the pQE9 vectordescribed supra, and then cloned into BamHI-HindIII sites of eukaryoticexpression vector pcDNA 3.1. Next, COS-7 cells were transfected withthis vector, by contacting cell samples with 150 ng of the plasmiddiscussed supra, and 150 ng of plasmid pcDNA 1 Amp, which containedeither cDNA for HLA-A2.1 or cDNA for HLA-A1, The well known DEAE-dextranchloroquine method was used. The cells were then incubated at 37° C.,for 48 hours, after which they were tested in a CTL stimulation assay.Specifically, the assay followed Traversari et al, Immunogenetics 35:145-148 (1992), incorporated by reference. In brief, 2500 CTLs,(NW38-IVS-1, see example 9, infra), in 100 ul RPMI supplemented with100% human serum, and 25 U/ml of recombinant IL-2 were added tomicrowells containing COS-7 transfectants (20,000 cells/well) After 24hours, 50 ul of supernatant were collected from each well, and TNF-αlevels were determined in a standard assay, i.e., one where cytotoxicityagainst WEHI 164 clone 13 cells were tested, using MTT. Positive cellswere used in the Western Blot analysis, described in the example whichfollows.

[0048] The CTLs used were CTL NW38-IVS-1, prepared in accordance withKnuth et al., Proc. Natl. Acad. Sci. USA 81: 3511-3515 (1984),incorporated by reference. Specifically, mixed lymphocyte T cellcultures were set up, by combining 10⁵ autologous NW38 MEL-1 tumorcells, and 10⁶ peripheral blood lymphocytes, taken from the subject. Thecytokine IL-2 was added, and the mixed culture was incubated for oneweek at 37° C. Tumor cells were removed, and a new aliquot of 5×10⁴tumor cells were added together with IL-2. This process was repeatedweekly, until a strong response was seen when tested against ⁵¹Crlabelled NW-MEL-38 cells. The responder T cells were collected andfrozen until used in further experiments.

EXAMPLE 8

[0049] Western Blot analysis was then carried out, using the serumsamples described supra, as well as cell lysates taken from the cellline NW-MEL-38, described supra, and the COS-7 transfectants, describedsupra, and the purified recombinant protein, also described supra. Serumsamples were taken from various points of the patient's therapy. Therewas no difference in the results.

[0050] In these assays, 1 ug of recombinant NY-ESO-1 protein, or 5 ul ofcell lysates of either type were diluted in SDS and boiled for fiveminutes, and then electrophoresed on a 15% SDS gel. After overnightblotting on nitrocellulose (0.45 um), and blocking with 3% BSA, theblots were incubated with serum, diluted at 1:1000, 1:10,000, and1:100,000, or with a monoclonal antibody against NY-ESO-1, diluted to1:50, as a positive control. The monoclonal antibody was prepared viaChen, et al., Proc. Natl. Acad. Sci. USA 5915-5919 (1996), incorporatedby reference and elaborated as follows. BALB/C mice were immunized viafive subcutaneous injections of recombinant NY-ESO-1 protein, at 2-3week intervals. The immunizing formulation included 50 ug of recombinantprotein in adjuvant. The first injection used Complete Freund'sAdjuvant, and Incomplete Freund's Adjuvant was used thereafter. Spleencells were taken from the immunized mice, and fused with mouse myelomacell line SP2/0, to generate hybridomas. Representative hybridoma E978was used for generation of mAbs.

[0051] Once hybridomas were generated, they were cloned, and theirsupernatants were screened against recombinant protein, using a standardsolid phase ELISA on microtiter plates. The assay was in accordance withDippold et al., Proc. Natl. Acad. Sci. USA 77: 6114-6118 (1980),incorporated by reference. A series of negative controls were also run,using recombinant NY-ESO-1. Serum antibodies which bound to recombinantprotein, produced by E. coli as described, supra were visualized usinggoat anti-human IgG, labelled with alkaline phosphatase at 1:10,000dilution, and were then visualized with NBT-phosphate. UntransfectedCOS-7 cells were also used as a control. Serum from a healthy individualwas also used as a control.

[0052] Strong reactivity against the recombinant protein was found atserum dilutions down to 1:100,000, and there was also reactivity againstlysate of NW-MEL-38. There was no reactivity found against theuntransfected COS-7 cells, nor did the serum from a healthy individualshow reactivity.

EXAMPLE 9

[0053] Four different forms of NY-ESO-1 are described supra, i.e., theform produced by SEQ ID NO: 1 in E. coli, as well as one consisting ofamino acids 10-180, one consisting of amino acids 10-121, and a form,expressed in the baculovirus vector system discussed supra whichconsisted of amino acids 10-180. Each form was used in ELISAs, followingthe above described protocols. All forms of the protein were found to beequally reactive with antibodies taken from various patients, as well asthe murine monoclonal antibodies discussed, supra.

EXAMPLE 10

[0054] In the testing of the COS-7 transfectants, supra, and the assaysdiscussed in this example, a cytolytic T cell line “NW38-IVS-1” wasused. This “CTL” was generated, via in vitro stimulation of theperipheral blood lymphocytes mentioned supra, using the tumor cell lineNW-MEL-38. This was done using standard techniques.

[0055] The CTL was used in a cytotoxicity assay with NW-MEL-38 (whichwas HLA-A1, A2 positive, and NY-ESO-1 positive), along with twoallogeneic cell lines which were NY-ESO-1 and HLA-A2 positive (SK-MEL-37and MZ-MEL-19), a cell line which is MHC Class I negative (SK-MEL-19), acell line which is HLA-A2 positive, but NY-ESO-1 negative (NW-MEL-145),along with control cell lines K562 and autologous phytohemagglutininstimulated blasts. Various effector/target ratios were used, and lysisof ⁵¹Cr labelled target cells was the parameter measured. FIG. 5 showsthis.

[0056] The results indicated that the CTL NW38-IVS-1 lysed both theautologous cell line NW MEL-38, and the allogeneic cell lines which wereHLA-A2 and ESO-1 positive. Hence, the CTL was reactive with allogeneicmaterials. See FIG. 6.

EXAMPLE 11

[0057] As patient NW38 was HLA-A1 and HLA-A2 positive, experiments werecarried out to determine which MHC molecule was the presenting molecule.

[0058] The same experiment, described supra with COS-7 cells was carriedout, except that, in these experiments, care was taken to secureseparate groups of cotransformants which had been transformed witheither HLA-A1 cDNA, or HLA-A2 cDNA, but not both. These results showthat the CTL NW38-IVS-1 lysed COS-7 transfectants containing bothNY-ESO-1 and HLA-A2 exclusively. See FIG. 6. The work also confirmed thespecificity of the CTL, since the NY-ESO-1 negative, HLA-A2 positivecells described in Example 9 were positive for other molecules known tobe processed to peptides presented by HLA-A2 molecules.

EXAMPLE 12

[0059] Once the presenting MHC molecule was identified as HLA-A2, ascreening of the amino acid sequence for NY-ESO-1 was carried out, toidentify all peptides which satisfy this motif, using the model setforth by D'Amaro et al., Human Immunol. 43: 13-18 (1995), and Drijfhout,et al., Human Immunol. 43: 1-12 (1995) incorporated by reference.Peptides corresponding to all of the amino acid sequences deducedthereby were synthesized, using standard techniques, and were then usedin cytotoxicity assays, following Knuth et al., Proc. Natl. Acad. Sci.USA 81: 3511-3515 (1984), incorporated by reference. Specifically, cellline CEMX721.174.T2 (“T2” hereafter), was used, because it does notprocess antigens to MHC complexed peptides, thereby making it ideal forexperiments of the type described herein. Samples of T2 cells werelabelled with 100 uCi of Na(⁵¹Cr)O₄, using standard methods, and werethen washed three times, followed by incubation with 10 ug/ml peptideand 2.5 ug/ml of β2-microglobulin. Incubation was for one hour, at roomtemperature. Then responder cells (100 ul of a suspension of CTLNW38-IVS-1) were added, at an effector/target ratio of 90:1, andincubated for four hours in a water saturated atmosphere, with 5% CO₂,at 37° C. Then, plates were centrifuged at 200×g for five minutes, 100ul of supernatant was removed, and radioactivity was measured. Thepercentage of ⁵¹Cr release was determined in accordance with knownstrategies. it was found that the peptides SLLMWITQCFL (SEQ ID NO: 4),SLLMWITQC (SEQ ID NO: 5), and QLSLLMWIT (SEQ ID NO: 6), were the threebest stimulators of CTLs. Comparable results were found when NW-MEL-38and cell lines SK-MEL-37 and MZ-MEL-19 were used as targets, as isshown, supra.

EXAMPLE 13

[0060] The amino acid sequence of the protein encoded by SEQ ID NO: 1was analyzed for peptide sequences which correspond to HLA bindingmotifs. This was done using the algorithm taught by Parker et al., J.Immunol. 142: 163 (1994), incorporated by reference. In the Table whichfollows, the amino acid sequence, the HLA molecule to which itpresumably binds, and the positions in SEQ ID NO: 1 are given. Theresulting complexes should provoke a cytolytic T cell response. Thiscould be determined by one skilled in the art following methods taughtby, e.g., van der Bruggen, et al., J. Eur. J. Immunol. 24: 3038-3043(1994), incorporated by reference. MHC/HLA Sequence Molecule PositionsGPESRLLEF HLA-A1 82-90 LLMWTTQCF HLA-A3 158-166 LMWITQCFL HLA-A3 159-167EPTVSGNIL HLA-A24 125-133 LQLSISACL HLA-A24 145-153 GARGPESRL HLA-B779-87 APRGPHGGA HLA-B7 60-68 ESRLLEFYL HLA-B7 84-92 APPLPVPGV HLA-B7113-121 FATPMEAEL HLA-B7  96-104 AADHRQLQL HLA-B7 139-147 GARGPESRLHLA-B8 79-87 ESRLLEFYL HLA-B8 84-92 VPGVLLKEF HLA-B35 118-126 ESRLLEFYLHLA-B35 84-92 GARGPESRL HLA-B35 79-87 LEFYLAMPF HLA-B44 88-96 PESRLLEFYHLA-B44 83-91 AELARRSLA HLA-B44 102-110 MEAELARRS HLA-B44 100-108QQLSLLMWI HLA-B52 154-162 AQDAPPLPV HLA-B52 110-110 LQLSISSCL HLA-B52145-153 ITQCFLPVF HLA-B52 162-170 LLEPYLAMPF HLA-A1 87-96 GPESRLLEFYHLA-A1 82-91 PLPVPGVLLK HLA-A3 115-124 RSLAQDAPPL HLA-A24 107-116APPLIPVPGVL HLA-B7 113-122 GARGPESRLL HLA-B7 79-88 GPHGGAASLG HLA-B763-72 APRGPHGGAA HLA-B7 60-69 GPRGAGAARA HLA-B7 44-53 TAADHRQLQL HLA-B8138-147 APPLPVPGVL HLA-B52 113-122 QQLSLLMWIT HLA-B52 154-163LQQLSLTJMWT HLA-B52 153-162 KEFTVSFNIL HLA-B52 124-133

[0061] The foregoing examples describe the isolation of a nucleic acidmolecule which encodes an esophageal cancer associated antigen.“Associated” is used herein because while it is clear that the relevantmolecule was expressed by esophageal cancer, other cancers, such asmelanoma, breast, prostate and lung also express the antigen.

[0062] The invention relates to those nucleic acid molecules whichencode antigens as described, and which hybridize to reference sequenceSEQ ID NO: 1 under stringent conditions. “Stringent conditions” as usedherein refers to conditions such as those specified in U.S. Pat. No.5,342,774, i.e., 18 hours of hybridization at 65° C., followed by fourone hour washes at 2×SSC, 0.1% SDS, and a final wash at 0.2×SSC, morepreferably 0.1×SSC, 0.1% SDS for 30 minutes, as well as alternateconditions which afford the same level of stringency, and more stringentconditions.

[0063] Also a part of the invention are expression vectors whichincorporate the nucleic acid molecules of the invention, in operablelinkage (i.e., “operably linked”) to a promoter. Construction of suchvectors is well within the skill of the art, as is the transformation ortransfection of cells, to produce eukaryotic cell lines, or prokaryoticcell strains which encode the molecule of interest. Exemplary of thehost cells which can be employed in this fashion are COS cells, CHOcells, yeast cells, insect cells (e.g., Spodoptera frugiperda), NIH 3T3cells, and so forth. Prokaryotic cells, such as E. coli and otherbacteria may also be used.

[0064] Also a part of the invention is the antigen described herein,both in original peptide form and in post translational modified form,as well as proteins consisting of at least amino acids 10-121, and nomore than 10-180, of the protein encoded by SEQ ID NO: 1. The moleculeis large enough to be antigenic without any posttranslationalmodification, and hence it is useful as an immunogen, when combined withan adjuvant (or without it), in both precursor and post-translationallymodified forms. These proteins can be used to determine whether or notantibodies are present in a sample, such as serum or blood, as shownsupra. The antibodies produced using this antigen, both poly andmonoclonal, are also a part of the invention as well as hybridomas whichmake the monoclonal antibody. These can be used therapeutically ordiagnostically as the whole molecule or in portions, as discussed infra.Also a part of the invention are reactive fragments, such as Fab,F(ab)₂′ and other fragments, as well as chimeras, humanized antibodies,recombinantly produced antibodies, and so forth. Especially preferredare chimeras where the entire antibody but the complementarilydetermining regions “CDRS” is human, but the CDRs are murine.

[0065] As is clear from the disclosure, one may use the proteins andnucleic acid molecules of the invention diagnostically. The SEREXmethodology discussed herein is premised on an immune response to apathology associated antigen. Hence, one may assay for the relevantpathology via, e.g., testing a body fluid sample of a subject, such asserum, for reactivity with the antigen per se. Reactivity would bedeemed indicative of possible presence of the pathology so, too, couldone assay for the expression of the antigen via any of the standardnucleic acid hybridization assays which are well known to the art, andneed not be elaborated upon herein. One could assay for antibodiesagainst the subject molecule, using standard immunoassays as well.

[0066] Analysis of SEQ ID NO: 1 will show that there are 5′ and 3′ noncoding regions presented therein. The invention relates to thoseisolated nucleic acid molecules which contain at least the codingsegment, i.e., nucleotides 54-593, and which may contain any or all ofnucleotides 1-53 and/or 594-747 of SEQ ID NO: 1.

[0067] Further analysis, as discussed supra, reveals that the moleculeis processed to peptides which provoke lysis by cytolytic T cells.Example 7 showed how this type of motif analysis can be carried out forHLA-A2 molecules. There has been a great deal of work in motifs forvarious MHC or HLA molecules, which is applicable here. Hence, a furtheraspect of the invention is a therapeutic method, wherein one or morepeptides which bind to an HLA molecule on the surface of a patient'stumor cells are administered to the patient, in an amount sufficient forthe peptides to bind to the MHC/HLA molecules, and provoke lysis by Tcells. The exemplification given supra for HLA-A2 molecules is by nomeans the only type of this administration that can be used. Anycombination of peptides may be used, such as those for other HLAmoleucles, described supra. These peptides, which may be used alone orin combination, as well as the entire protein or immunoreactive portionsthereof, may be administered to a subject in need thereof, using any ofthe standard types of administration, such as intravenous, intradermal,subcutaneous, oral, rectal, and transdermal administration. Standardpharmaceutical carriers, adjuvants, such as saponins, GM-CSF, andinterleukins and so forth may also be used. Further, these peptides andproteins may be formulated into vaccines with the listed material, asmay dendritic cells, or other cells which present relevant MHC/peptidecomplexes. These peptides may also be used to form multimeric complexesof HLA/peptides, such as those described by Dunbar, et al., Curr. Biol.8: 413-416 (1998), incorporated by reference, wherein fourpeptide/MHC/biotin complexes are attached to a streptavidin or avidinmolecule. Such complexes can be used to identify and/or to stimulate Tcell precursors.

[0068] Similarly, the invention contemplates therapies wherein thenucleic acid molecule which encodes NY-ESO-1 is incorporated into avector, such as an adenovirus based vector, to render it transfectableinto eukaryotic cells, such as human cells. Similarly, nucleic acidmolecules which encode one or more of the peptides may be incorporatedinto these vectors, which are then the major constituent of nucleic acidbases therapies.

[0069] Any of these assays can also be used in progression/regressionstudies. One can monitor the course of abnormality involving expressionof NY-ESO-1, simply by monitoring levels of the protein, its expression,and so forth using any or all of the methods set forth supra.

[0070] It should be clear that these methodologies may also be used totrack the efficacy of a therapeutic regime. Essentially, one can take abaseline value for the NY-ESO-1 protein, using any of the assaysdiscussed supra, administer a given therapeutic agent, and then monitorlevels of the protein thereafter, observing changes in ESO-1 levels asindicia of the efficacy of the regime.

[0071] As was indicated supra, the invention involves, inter alia, therecognition of an “integrated” immune response to the NY-ESO molecule.One ramification of this is the ability to monitor the course of cancertherapy. In this method, which is a part of the invention, a subject inneed of the therapy receives a vaccination of a type described herein.Such a vaccination results, e.g., in a T cell response against cellspresenting HLA/peptide complexes on their cells. The response alsoincludes an antibody response, possibly a result of the release ofantibody provoking proteins via the lysis of cells by the T cells.Hence, one can monitor the effect of a vaccine, by monitoring an immuneresponse. As is indicated, supra, an increase in antibody titer or Tcell count may be taken as an indicia of progress with a vaccine, andvice versa. Hence, a further aspect of the invention is a method formonitoring efficacy of a vaccine, following administration thereof, bydetermining levels of antibodies in the subject which are specific forthe vaccine itself, or a large molecules of which the vaccine is a part.

[0072] The effects of a vaccine can also be measured by monitoring the Tcell-response of the subject receiving the vaccine. A number of assayscan be used to measure the precursor frequency of these in vitrostimulated T cells. These include, but are not limited to, chromiumrelease assays, TNF release assays, IFNγ release assays, an ELISPOTassay, and so forth. Changes in precursor T cell frequences can bemeasured and correlated to the efficacy of the vaccine. Additionalmethods which can be employed include the use of multimeric complexes ofMHC/peptides. An example of such complexes is the tetramericHLA/peptide-biotin-streptavidin system of Dunbar, et al. Curr. Biol. 8:413-416 (1998), incorporated by reference.

[0073] The identification of NY-ESO-1 proteins as being implicated inpathological conditions such as cancer also suggests a number oftherapeutic approaches in addition to those discussed supra. Theexperiments set forth supra establish that antibodies are produced inresponse to expression of the protein. Hence, a further embodiment ofthe invention is the treatment of conditions which are characterized byaberrant or abnormal levels of NY-ESO-1 proteins, via administration ofantibodies, such as humanized antibodies, antibody fragments, and soforth. These may be tagged or labelled with appropriate cystostatic orcytotoxic reagents.

[0074] T cells may also be administered. It is to be noted that the Tcells may be elicited in vitro using immune responsive cells such asdendritic cells, lymphocytes, or any other immune responsive cells, andthen reperfused into the subject being treated.

[0075] Note that the generation of T cells and/or antibodies can also beaccomplished by administering cells, preferably treated to be renderednon-proliferative, which present relevant T cell or B cell epitopes forresponse, such as the epitopes discussed supra.

[0076] The therapeutic approaches may also include antisense therapies,wherein an antisense molecule, preferably from 10 to 100 nucleotides inlength, is administered to the subject either “neat” or in a carrier,such as a liposome, to facilitate incorporation into a cell, followed byinhibition of expression of the protein. Such antisense sequences mayalso be incorporated into appropriate vaccines, such as in viral vectors(e.g., Vaccinia), bacterial constructs, such as variants of the knownBCG vaccine, and so forth.

[0077] Also a part of the inventions are peptides, which can benonamers, decamers, or undecamers defined as having a core sequence:

[0078] LLMWIT (SEQ ID NO: 7)

[0079] which have at least one additional residue terminal to the firstL residue, preferably serine and may have as many as three, whereinSerine is linked to L to form -SL-, and 0-4 additional amino acids atthe C-terminus which, as shown supra, bind to HLA-A2 molecules, therebyprovoking a CTL response. These peptides may be used therapeutically,via administration to a patient who is HLA-A2 positive, and expressesNY-ESO-1 in connection with a pathology, as well as diagnostically,i.e., to determine if HLA-A2 positive cells are present, or if relevantCTLs are present, and so forth.

[0080] The HLA-A2 molecule is an MHC Class I molecule, and T cells whichrespond to complexes of peptides and class I molecules are generallyCD8⁺ cells. Another subset of T cells, CD4⁺ cells, responds to complexesof MHC-Class II molecules and peptides, and MHC-Class II restricted CD4⁺T cell responses against recombinant NY-ESO-1, presented by autologouscultured dendritic cells have been detected in melanoma patients.Specifically, in results not described herein, CD4⁺ cells were separatedfrom other cells from PBLs or serum samples, using well knowntechniques. Then, they were admixed with dendritic cells which had beenpulsed with NY-ESO-1 protein. Proliferation of CD4⁺ cells was observed,bringing another facet to the integrated immune response discussedherein. Hence, a further aspect of this invention are these CD4⁺ Tcells, peptides which bind to the MHC-Class II molecules, and their usein therapy.

[0081] Other features and applications of the invention will be clear tothe skilled artisan, and need not be set forth herein.

[0082] The terms and expression which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1 8 752 base pairs nucleic acid single linear 1 atcctcgtgg gccctgaccttctctctgag agccgggcag aggctccgga gccatgcagg 60 ccgaaggccg gggcacagggggttcgacgg gcgatgctga tggcccagga ggccctggca 120 ttcctgatgg cccagggggcaatgctggcg gcccaggaga ggcgggtgcc acgggcggca 180 gaggtccccg gggcgcaggggcagcaaggg cctcggggcc gggaggaggc gccccgcggg 240 gtccgcatgg cggcgcggcttcagggctga atggatgctg cagatgcggg gccagggggc 300 cggagagccg cctgcttgagttctacctcg ccatgccttt cgcgacaccc atggaagcag 360 agctggcccg caggagcctggcccaggatg ccccaccgct tcccgtgcca ggggtgcttc 420 tgaaggagtt cactgtgtccggcaacatac tgactatccg actgactgct gcagaccacc 480 gccaactgca gctctccatcagctcctgtc tccagcagct ttccctgttg atgtggatca 540 cgcagtgctt tctgcccgtgtttttggctc agcctccctc agggcagagg cgctaagccc 600 agcctggcgc cccttcctaggtcatgcctc ctcccctagg gaatggtccc agcacgagtg 660 gccagttcat tgtgggggcctgattgtttg tcgctggagg aggacggctt acatgtttgt 720 ttctgtagaa aataaaactgagctacgaaa aa 752 31 base pairs nucleic acid single linear 2 CACACAGGATCCATGGATGC TGCAGATGCG G 31 32 base pairs nucleic acid single linear 3CACACAAAGC TTGGCTTAGC GCCTCTGCCC TG 32 11 amino acids amino acid linear4 Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu 5 10 9 amino acids aminoacid linear 5 Ser Leu Leu Met Trp Ile Thr Gln Cys 5 9 amino acids aminoacid linear 6 Gln Leu Ser Leu Leu Met Trp Ile Thr 5 6amino acids aminoacid linear 7 Leu Leu Met Trp Ile Thr 5 180 amino acid linear 8 Met GlnAla Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 5 10 15 Gly ProGly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30 Gly ProGly Glu Ala Gly Ala Thr Gly Gly Arg Ala Pro Arg Gly Ala 35 40 45 Gly AlaAla Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60 His GlyGly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala 65 70 75 80 ArgGly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95 AlaThr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120125 Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130135 140 Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met145 150 155 160 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln ProPro Ser 165 170 175 Gly Gln Arg Arg 180

We claim:
 1. An isolated antibody or binding fragment of an antibodywhich binds with a protein which is encoded by an isolated nucleic acidmolecule, the complementary sequence of which hybridizes, understringent conditions, to a nucleic acid molecule comprising nucleotides54-593 of SEQ ID NO:
 1. 2. The isolated antibody of claim 1, whereinsaid antibody is a monoclonal antibody.
 3. The isolated antibody ofclaim 1, wherein said antibody is a chimeric antibody.
 4. The isolatedantibody of claim 3, wherein said chimeric antibody comprises ahumanized antibody with a murine CDR region.
 5. Hybridoma cell linewhich produces the monoclonal antibody of claim
 2. 6. A method forscreening for cancer in a sample, comprising contacting said sample withthe isolated antibody of claim 1, and determining binding of saidantibody to a target as an indicator of cancer.
 7. The method of claim6, wherein said cancer is melanoma, breast cancer, prostate cancer, lungcancer, hepatoma, ovarian cancer, thyroid cancer, bladder cancer, orlymphoma.
 8. A method for determining presence of antibodies against acancer associated antigen in a sample, comprising contacting said samplewith an isolated protein encoded by a nucleic acid molecule whichcomprises nucleotide 54-593 of SEQ ID NO: 1, and determining binding ofantibodies thereto as a determination of antibodies against a cancerassociated antigen in said sample.
 9. A method for determiningregression, progression of onset of a cancerous condition comprisingmonitoring a sample from a patient with said cancerous condition for aparameter selected from the group consisting of (i) NY-ESO-1 protein,and (ii) a peptide derived from NY-ESO-1 protein, with an antibody whichbinds to (i) or (ii), wherein amount of said parameter is indicative ofprogression or regression or onset of said cancerous condition.
 10. Themethod of claim 9, wherein said sample is a body fluid or exudate. 11.The method of claim 9, wherein said sample is a tissue.
 12. The methodof claim 11, wherein said antibody is labelled with a radioactive labelor an enzyme.
 13. The method of claim 11, wherein said antibody is amonoclonal antibody.
 14. A method for treating a subject afflicted witha cancerous condition comprising administering to said subject anantibody which specifically binds to NY-ESO-1 protein or to an ESO-1derived peptide expressed on a cancerous cell associated with saidcancerous condition, said antibody being coupled to an anticancer agent,in an amount sufficient to treat said cancerous condition.
 15. A methodfor treating a subject afflicted with a cancerous condition comprisingadministering to said subject an antibody which specifically binds toNY-ESO-1, said antibody being coupled to an anticancer agent, in anamount sufficient to treat said cancerous condition.
 16. Isolatedprotein consisting of at least amino acids 10-121 and no more than aminoacids 10-180 of the protein encoded by the isolated nucleic acidmolecule having the nucleotide sequence set forth in SEQ ID NO:
 1. 17.The isolated protein of claim 16, consisting of amino acids 10-121. 18.The isolated protein of claim 16, consisting of amino acids 10-180. 19.The isolated protein of claim 16, wherein said protein is glycosylated.20. Immunogenic composition comprising the isolated protein of claim 16,and a carrier.
 21. A method for determining presents of antibodiesagainst a cancer associated antigen in a sample, comprising contactingsaid sample with the isolated protein of claim 16, and determiningbinding to said protein as a determination of antibodies to a cancerassociated antigen in said sample.